Maize event dp-915635-4 and methods for detection thereof

ABSTRACT

Embodiments disclosed herein relate to the field of plant molecular biology, specifically to DNA constructs for conferring insect resistance to a plant. Embodiments disclosed herein relate to insect resistant corn plant containing event DP-915635-4, and to assays for detecting the presence of event DP-915635-4 in samples and compositions thereof

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“8425_SeqList.txt” created on Nov. 13, 2020 and having a size of 202kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD

Embodiments disclosed herein relate to the field of plant molecularbiology, including to DNA constructs for conferring insect resistance toa plant. Embodiments disclosed herein also include insect resistant cornplant containing event DP-915635-4 and assays for detecting the presenceof event DP-915635-4 in a sample and compositions thereof.

BACKGROUND

Corn is an important crop and is a primary food source in many areas ofthe world. Damage caused by insect pests is a major factor in the lossof the world's corn crops, despite the use of protective measures suchas chemical pesticides. In view of this, insect resistance has beengenetically engineered into crops such as corn in order to controlinsect damage and to reduce the need for traditional chemicalpesticides. One group of genes which have been utilized for theproduction of transgenic insect resistant crops is the delta-endotoxingroup from Bacillus thuringiensis (Bt). Delta-endotoxins have beensuccessfully expressed in crop plants such as cotton, potatoes, rice,sunflower, as well as corn, and in certain circumstances have proven toprovide excellent control over insect pests. (Perlak, F. J et al. (1990)Bio/Technology 8:939-943; Perlak, F. J. et al. (1993) Plant Mol. Biol.22:313-321; Fujimoto, H. et al. (1993) Bio/Technology 11:1151-1155; Tuet al. (2000) Nature Biotechnology 18:1101-1104; PCT publication WO01/13731; and Bing, J. W. et al. (2000) Efficacy of Cry 1F TransgenicMaize, 14^(th) Biennial International Plant Resistance to InsectsWorkshop, Fort Collins, Colo.).

The expression of transgenes in plants is known to be influenced by manydifferent factors, including the orientation and composition of thecassettes driving expression of the individual genes of interest, andthe location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising et al.(1988) Ann. Rev. Genet. 22:421-477).

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest.

It is possible to detect the presence of a transgene by a nucleic aciddetection method by, e.g., a polymerase chain reaction (PCR) or DNAhybridization using nucleic acid probes. These detection methodsgenerally focus on frequently used genetic elements, such as promoters,terminators, marker genes, etc., because for many DNA constructs, thecoding region is interchangeable. As a result, such methods may not beuseful for discriminating between different events, particularly thoseproduced using the same DNA construct or very similar constructs unlessthe DNA sequence of the flanking DNA adjacent to the insertedheterologous DNA is known

SUMMARY

The embodiments relate to the insect resistant corn (Zea mays) plantevent DP-915635-4 , also referred to as “maize line DP-915635-4 ,”“maize event DP-915635-4 ,” and “DP-915635-4 maize,” to the DNA plantexpression construct of corn plant event DP-915635-4 , and to methodsand compositions for the detection of the transgene construct, flanking,and insertion (the target locus) regions in corn plant event DP-915635-4and progeny thereof.

In one aspect compositions and methods relate to methods for producingand selecting an insect resistant monocot crop plant. Compositionsinclude a DNA construct that when expressed in plant cells and plantsconfers resistance to insects. In one aspect, a DNA construct, capableof introduction into and replication in a host cell, is provided thatwhen expressed in plant cells and plants confers insect resistance tothe plant cells and plants. Maize event DP-915635-4 was produced byAgrobacterium-mediated transformation with plasmid PHP83175. Asdescribed herein, these events include the IPD079Ea (polynucleotide SEQID NO: 4 and amino acid SEQ ID NO: 5) cassette (Table 1), which confersresistance to certain Coleopteran plant pests. The insect controlcomponents have demonstrated efficacy against Coleopteran insectspecies, particularly western corn rootworm (WCR).

A first cassette contains the insecticidal protein gene, IPD079Ea, fromOphioglossum pendulum (international patent application publicationnumber WO 2017023486). The expressed IPD079Ea protein in plants iseffective against certain coleopteran pests. The IPD079Ea protein is 479amino acids in length and has a molecular weight of approximately 52kDa. Expression of the IPD079Ea gene is controlled by three copies ofthe enhancer region, showing root-specific activity, from the Sorghum(Sorghum bicolor) root cortical RCc3 (sb-RCc3) gene (internationalpatent application publication number WO 2012112411) followed by thepromoter region upstream of a Zea mays PCO118362 mRNA sequence (zm PCOa)identified as having root-specific activity (international patentapplication publication number WO 2017222821) and the intron region fromthe Zea mays ortholog of a rice (Oryza sativa) hypothetical protein(zm-HPLV9) gene, a predicted Zea mays calmodulin 5 gene (Phytozome geneID Zm00008a029682; international patent application publication numberWO 2016109157). The terminator for the IPD079Ea gene is the terminatorregion from the Sorghum (Sorghum bicolor) subtilisin-chymotrypsininhibitor 1B (sb-SCI-1B) gene (international patent applicationpublication number WO 2018102131). Three additional terminators arepresent to prevent transcriptional interference: the terminator regionfrom the maize W64 line 27-kDa gamma zein (Z27G) gene (Das et al., 1991;Liu et al., 2016), the terminator region from the Arabidopsis thalianaubiquitin 14 (UBQ14) gene (Callis et al., 1995), and the terminatorregion from the maize In2-1 gene (Hershey and Stoner, 1991).

A second gene cassette (mo pat gene cassette) contains thephosphinothricin acetyl transferase gene (mo-pat) from Streptomycesviridochromogenes (Wohlleben et al., 1988). The mo-pat gene expressesthe phosphinothricin acetyl transferase (PAT) enzyme that conferstolerance to phosphinothricin. The PAT protein is 183 amino acids inlength and has a molecular weight of approximately 21 kDa. Expression ofthe mo-pat gene is controlled by the promoter and intron region of theOryza sativa (rice) actin (os-actin) gene (GenBank accession CP018159),in conjunction with a third copy of the CaMV35S terminator. Twoadditional terminators are present to prevent transcriptionalinterference: the terminator regions from the Sorghum bicolor (sorghum)ubiquitin (sb-ubi) gene (Phytozome gene ID Sobic.004G049900.1) andγ-kafarin (sb-gkaj) gene (de Freitas et al., 1994), respectively.

A third gene cassette (pmi gene cassette) contains the phosphomannoseisomerase (pmi) gene from Escherichia coli (Negrotto et al., 2000).Expression of the PMI protein in plants serves as a selectable markerwhich allows plant tissue growth with mannose as the carbon source. ThePMI protein is 391 amino acids in length and has a molecular weight ofapproximately 43 kDa. As present in the T-DNA region of PHP74643, thepmi gene lacks a promoter, but its location next to the flippaserecombination target site, FRT1, allows post-recombination expression byan appropriately-placed promoter. The terminator for the pmi gene is afourth copy of the pinII terminator. An additional Z19 terminatorpresent is intended to prevent transcriptional interference betweencassettes.

According to some embodiments, compositions and methods are provided foridentifying a novel corn plant designated DP-915635-4 (ATCC DepositNumber PTA-126746). The methods are based on primers or probes whichspecifically recognize 5′ and/or 3′ flanking sequence of DP-915635-4.DNA molecules are provided that comprise primer sequences that whenutilized in a PCR reaction will produce amplicons unique to thetransgenic event DP-915635-4. In one embodiment, the corn plant and seedcomprising these molecules is contemplated. Further, kits utilizingthese primer sequences for the identification of the DP-915635-4 eventare provided.

Some embodiments relate to specific flanking sequences of DP-915635-4 asdescribed herein, which can be used to develop identification methodsfor DP-915635-4 in biological samples. More particularly, the disclosurerelates to 5′ and/or 3′ flanking regions of DP-915635-4, which can beused for the development of specific primers and probes. Furtherembodiments relate to identification methods for the presence ofDP-915635-4 in biological samples based on the use of such specificprimers or probes.

According to some embodiments, methods of detecting the presence of DNAcorresponding to the corn event DP-915635-4 in a sample are provided.Such methods comprise: (a) contacting the sample comprising DNA with aDNA primer set, that when used in a nucleic acid amplification reactionwith genomic DNA extracted from corn comprising event DP-915635-4produces an amplicon that is diagnostic for corn event DP-915635-4,respectively; (b) performing a nucleic acid amplification reaction,thereby producing the amplicon; and (c) detecting the amplicon. In someaspects, the primer set comprises SEQ ID NOs: 6 and 7, and optionally aprobe comprising SEQ ID NO: 8.

According to some embodiments, methods of detecting the presence of aDNA molecule corresponding to the DP-915635-4 event in a samplecomprise: (a) contacting the sample comprising DNA extracted from a cornplant with a DNA probe molecule that hybridizes under stringenthybridization conditions with DNA extracted from corn event DP-915635-4and does not hybridize under the stringent hybridization conditions witha control corn plant DNA; (b) subjecting the sample and probe tostringent hybridization conditions; and (c) detecting hybridization ofthe probe to the DNA extracted from corn event DP-915635-4. Morespecifically, a method for detecting the presence of a DNA moleculecorresponding to the DP-915635-4 event in a sample consist of (a)contacting the sample comprising DNA extracted from a corn plant with aDNA probe molecule that comprises sequences that are unique to theevent, e.g. junction sequences, wherein said DNA probe moleculehybridizes under stringent hybridization conditions with DNA extractedfrom corn event DP-915635-4 and does not hybridize under the stringenthybridization conditions with a control corn plant DNA; (b) subjectingthe sample and probe to stringent hybridization conditions; and (c)detecting hybridization of the probe to the DNA.

In addition, a kit and methods for identifying event DP-915635-4 in abiological sample which detects a DP-915635-4 specific region areprovided.

DNA molecules are provided that comprise at least one junction sequenceof DP-915635-4; wherein a junction sequence spans the junction locatedbetween heterologous DNA inserted into the genome and the DNA from themaize cell flanking the insertion site and may be diagnostic for theDP-915635-4 event.

According to some embodiments, methods of producing an insect resistantcorn plant comprise the steps of: (a) sexually crossing a first parentalcorn line comprising the expression cassettes disclosed herein, whichconfer resistance to insects, and a second parental corn line that lackssuch expression cassettes, thereby producing a plurality of progenyplants; and (b) selecting a progeny plant that is insect resistant. Suchmethods may optionally comprise the further step of back-crossing theprogeny plant to the second parental corn line to produce atrue-breeding corn plant that is insect resistant.

Some embodiments provide a method of producing a corn plant that isresistant to insects comprising transforming a corn cell with the DNAconstruct PHP74643, growing the transformed corn cell into a corn plant,selecting the corn plant that shows resistance to insects, and furthergrowing the corn plant into a fertile corn plant. The fertile corn plantcan be self-pollinated or crossed with compatible corn varieties toproduce insect resistant progeny.

Some embodiments further relate to a DNA detection kit for identifyingmaize event DP-915635-4 in biological samples. The kit comprises a firstprimer which specifically recognizes the 5′ or 3′ flanking region ofDP-915635-4, and a second primer which specifically recognizes asequence within the non-native target locus DNA of DP-915635-4,respectively, or within the flanking DNA, for use in a PCRidentification protocol. A further embodiment relates to a kit foridentifying event DP-915635-4 in biological samples, which kit comprisesa specific probe having a sequence which corresponds or is complementaryto, a sequence having between about 80% and 100% sequence identity witha specific region of event DP-915635-4. The sequence of the probecorresponds to a specific region comprising part of the 5′ or 3′flanking region of event DP-915635-4. In some embodiments, the first orsecond primer comprises any one of SEQ ID NOs: 6-7, 9-10, 12-13, 15-16,or 18-19.

The methods and kits encompassed by the embodiments disclosed herein canbe used for different purposes such as, but not limited to thefollowing: to identify event DP-915635-4 in plants, plant material or inproducts such as, but not limited to, food or feed products (fresh orprocessed) comprising, or derived from plant material; additionally oralternatively, the methods and kits can be used to identify transgenicplant material for purposes of segregation between transgenic andnon-transgenic material; additionally or alternatively, the methods andkits can be used to determine the quality of plant material comprisingmaize event DP-915635-4. The kits may also contain the reagents andmaterials necessary for the performance of the detection method.

A further embodiment relates to the DP-915635-4 maize plant or itsparts, including, but not limited to, pollen, ovules, vegetative cells,the nuclei of pollen cells, and the nuclei of egg cells of the cornplant DP-915635-4 and the progeny derived thereof In another embodiment,the DNA primer molecules targeting the maize plant and seed ofDP-915635-4 provide a specific amplicon product

DESCRIPTION OF THE DRAWINGS

FIG. 1 . shows a schematic diagram of plasmid PHP83175 with geneticelements indicated. Plasmid size is 74,997 bp (SEQ ID NO: 1).

FIG. 2 . shows a schematic diagram of the T-DNA indicating six genecassettes. The T-DNA was used to transform a pre-characterized maizeline containing FRT1 and FRT6 sites. The region containing the pmi gene,the mo-pat gene, and the ipd079Ea gene between the FRT1 and FRT6 sitesin the T-DNA was integrated into the maize line in a site-specificmanner. The zm-wus2 and zm-odp2 developmental genes were present toincrease transformation efficiency. The zm-wus2, zm-odp2, and mo-Flpgenes were not incorporated in the final product.

FIG. 3 . shows a schematic map of the insertion in DP915635 maize basedon the SbS analysis described. The flanking maize genome (including thezm-SEQ158 and zm-SEQ159 regions) is represented by the horizontal blackbars. A single copy of the intended insertion, derived from PHP83175 andPHP73878, is integrated into the maize genome (SEQ ID NO: 2 is theinsert T-DNA sequence). Within the insertion, the landing pad sequencesfrom PHP73878 and the trait genes derived from PHP83175 are highlighted.SEQ ID NO: 3 is the complete insert sequence and flanking regions. TheFRT1 and FRT6 sites that are the targets of recombination during the SSIprocess are highlighted.

FIG. 4 . shows a schematic Diagram of the Transformation and Developmentof DP-915635-4.

FIG. 5 is a table showing hybrid performance of event DP-915635-4compared to a base entry for non-yield agronomic traits.

FIG. 6 is a table showing inbred performance of event DP-915635-4compared to a base entry for all agronomic traits.

DETAILED DESCRIPTION

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof, and so forth. All technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs unlessclearly indicated otherwise.

Compositions of this disclosure include seed deposited as ATCC PatentDeposit No. PTA-126746 and plants, plant cells, and seed derivedtherefrom. Applicant(s) deposited at least 2500 seeds of maize eventDP-915635-4 (Patent Deposit No. PTA-126746) with the American TypeCulture Collection (ATCC), Manassas, Va. 20110-2209 USA, on March 27,2020. These deposits will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. The seeds deposited with the ATCCon Mar. 27, 2020 were taken from the deposit maintained by PioneerHi-Bred International, Inc., 7250 NW 62^(nd) Avenue, Johnston, Iowa50131-1000. Access to this deposit will be available during the pendencyof the application to the Commissioner of Patents and Trademarks andpersons determined by the Commissioner to be entitled thereto uponrequest. Upon allowance of any claims in the application, theApplicant(s) will make available to the public, pursuant to 37 C.F.R. §1.808, sample(s) of the deposit of at least 625 seeds of hybrid maizewith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209. This deposit of seed of maize eventDP-915635-4 will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the enforceable life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.Additionally, Applicant(s) have satisfied all the requirements of 37C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant(s) have no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant(s) do not waiveany infringement of their rights granted under this patent or rightsapplicable to event DP-915635-4 under the Plant Variety Protection Act(7 USC 2321 et seq.). Unauthorized seed multiplication is prohibited.The seed may be regulated.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the terms “insect resistant” and “impacting insectpests” refers to effecting changes in insect feeding, growth, and/orbehavior at any stage of development, including but not limited to:killing the insect; retarding growth; reducing reproductive capability;inhibiting feeding; and the like.

As used herein, the terms “pesticidal activity” and “insecticidalactivity” are used synonymously to refer to activity of an organism or asubstance (such as, for example, a protein) that can be measured bynumerous parameters including, but not limited to, pest mortality, pestweight loss, pest attraction, pest repellency, and other behavioral andphysical changes of a pest after feeding on and/or exposure to theorganism or substance for an appropriate length of time. For example,“pesticidal proteins” are proteins that display pesticidal activity bythemselves or in combination with other proteins.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can exist of either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process which is extraneous to the original insertDNA molecule, e.g. fragments associated with the transformation event. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 10 bp (in some narrower embodiments, at least 20bp, at least 50 bp, and up to at least 5000 bp), which is located eitherimmediately upstream of and contiguous with and/or immediatelydownstream of and contiguous with the original non-native insert DNAmolecule. Transformation procedures of the foreign DNA may result intransformants containing different flanking regions characteristic andunique for each transformant. When recombinant DNA is introduced into aplant through traditional crossing, its flanking regions will generallynot be changed. It may be possible for single nucleotide changes tooccur in the flanking regions through generations of plant breeding andtraditional crossing. Transformants will also contain unique junctionsbetween a piece of heterologous insert DNA and genomic DNA, or two (2)pieces of genomic DNA, or two (2) pieces of heterologous DNA. A“junction” is a point where two (2) specific DNA fragments join. Forexample, a junction exists where insert DNA joins flanking DNA. Ajunction point also exists in a transformed organism where two (2) DNAfragments join together in a manner that is modified from that found inthe native organism. “Junction DNA” refers to DNA that comprises ajunction point. Junction sequences set forth in this disclosure includea junction point located between the maize genomic DNA and the 5′ end ofthe insert, which range from at least −5 to +5 nucleotides of thejunction point (SEQ ID NO: 26), from at least −10 to +10 nucleotides ofthe junction point (SEQ ID NO: 27), and from at least −25 to +25nucleotides of the junction point (SEQ ID NO: 28); and a junction pointlocated between the 3′ end of the insert and maize genomic DNA, whichrange from at least −5 to +5 nucleotides of the junction point (SEQ IDNO: 29), from at least −10 to +10 nucleotides of the junction point (SEQID NO: 30), and from at least −25 to +25 nucleotides of the junctionpoint (SEQ ID NO: 31). Junction sequences set forth in this disclosurealso include a junction point located between the target locus and the5′ end of the insert. In some embodiments, SEQ ID NOs: 8 or 21 forDP-915635-4 represent the junction point located between the targetlocus and the 5′ end of the insert. The complete insert with flankingregions is represented in SEQ ID NO: 3.

As used herein, “heterologous” in reference to a nucleic acid sequenceis a nucleic acid sequence that originates from a different non-sexuallycompatible species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. For example, a promoter operably linkedto a heterologous nucleotide sequence can be from a species differentfrom that from which the nucleotide sequence was derived, or, if fromthe same species, the promoter is not naturally found operably linked tothe nucleotide sequence. A heterologous protein may originate from aforeign species, or, if from the same species, is substantially modifiedfrom its original form by deliberate human intervention.

The term “regulatory element” refers to a nucleic acid molecule havinggene regulatory activity, i.e. one that has the ability to affect thetranscriptional and/or translational expression pattern of an operablylinked transcribable polynucleotide. The term “gene regulatory activity”thus refers to the ability to affect the expression of an operablylinked transcribable polynucleotide molecule by affecting thetranscription and/or translation of that operably linked transcribablepolynucleotide molecule. Gene regulatory activity may be positive and/ornegative and the effect may be characterized by its temporal, spatial,developmental, tissue, environmental, physiological, pathological, cellcycle, and/or chemically responsive qualities as well as by quantitativeor qualitative indications.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequencecomprises proximal and more distal upstream elements, the latterelements are often referred to as enhancers. Accordingly, an “enhancer”is a nucleotide sequence that can stimulate promoter activity and may bean innate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different regulatory elements may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters that cause a nucleic acid fragment to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical or similar promoter activity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect numerous parameters including, processing of theprimary transcript to mRNA, mRNA stability and/or translationefficiency.

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

A DNA construct is an assembly of DNA molecules linked together thatprovide one or more expression cassettes. The DNA construct may be aplasmid that is enabled for self-replication in a bacterial cell andcontains various endonuclease enzyme restriction sites that are usefulfor introducing DNA molecules that provide functional genetic elements,i.e., promoters, introns, leaders, coding sequences, 3′ terminationregions, among others; or a DNA construct may be a linear assembly ofDNA molecules, such as an expression cassette. The expression cassettecontained within a DNA construct comprises the necessary geneticelements to provide transcription of a messenger RNA. The expressioncassette can be designed to express in prokaryotic cells or eukaryoticcells. Expression cassettes of the embodiments are designed to expressin plant cells.

The DNA molecules disclosed herein are provided in expression cassettesfor expression in an organism of interest. The cassette includes 5′ and3′ regulatory sequences operably linked to a coding sequence. “Operablylinked” means that the nucleic acid sequences being linked arecontiguous and, where necessary to join two protein coding regions,contiguous and in the same reading frame. Operably linked is intended toindicate a functional linkage between a promoter and a second sequence,wherein the promoter sequence initiates and mediates transcription ofthe DNA sequence corresponding to the second sequence. The cassette mayadditionally contain at least one additional gene to be co-transformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes or multiple DNA constructs.

The expression cassette may include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region, acoding region, and a transcriptional and translational terminationregion functional in the organism serving as a host. The transcriptionalinitiation region (e.g., the promoter) may be native or analogous, orforeign or heterologous to the host organism. Additionally, the promotermay be the natural sequence or alternatively a synthetic sequence. Theexpression cassettes may additionally contain 5′ leader sequences in theexpression cassette construct. Such leader sequences can act to enhancetranslation.

It is to be understood that as used herein the term “transgenic”generally includes any cell, cell line, callus, tissue, plant part, orplant, the genotype of which has been altered by the presence of aheterologous nucleic acid including those initially so altered as wellas those created by sexual crosses or asexual propagation from theinitial transgenic and retains such heterologous nucleic acids.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct(s), including a nucleic acid expressioncassette that comprises a transgene of interest, the regeneration of apopulation of plants resulting from the insertion of the transgene intothe genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. An eventis characterized phenotypically by the expression of the transgene. Atthe genetic level, an event is part of the genetic makeup of a plant.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another variety, wherein the progenyincludes the heterologous DNA. After back-crossing to a recurrentparent, the inserted DNA and the linked flanking genomic DNA from thetransformed parent is present in the progeny of the cross at the samechromosomal location. A progeny plant may contain sequence changes tothe insert arising as a result of conventional breeding techniques. Theterm “event” also refers to DNA from the original transformantcomprising the inserted DNA and flanking sequence immediately adjacentto the inserted DNA that would be expected to be transferred to aprogeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

An insect resistant DP-915635-4 corn plant may be bred by first sexuallycrossing a first parental corn plant having the transgenic DP-915635-4event plant and progeny thereof derived from transformation with theexpression cassettes of the embodiments that confers insect resistance,and a second parental corn plant that lacks such expression cassettes,thereby producing a plurality of first progeny plants; and thenselecting a first progeny plant that is resistant to insects; andselfing the first progeny plant, thereby producing a plurality of secondprogeny plants; and then selecting from the second progeny plants aninsect resistant plant. These steps can further include theback-crossing of the first insect resistant progeny plant or the secondinsect resistant progeny plant to the second parental corn plant or athird parental corn plant, thereby producing a corn plant that isresistant to insects. The term “selfing” refers to self-pollination,including the union of gametes and/or nuclei from the same organism.

As used herein, the term “plant” includes reference to whole plants,parts of plants, plant organs (e.g., leaves, stems, roots, etc.), seeds,plant cells, and progeny of same. In some embodiments, parts oftransgenic plants comprise, for example, plant cells, protoplasts,tissues, callus, embryos as well as flowers, stems, fruits, leaves, androots originating in transgenic plants or their progeny previouslytransformed with a DNA molecule disclosed herein, and thereforeconsisting at least in part of transgenic cells.

As used herein, the term “plant cell” includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that may be used is generally as broadas the class of higher plants amenable to transformation techniques,including both monocotyledonous and dicotyledonous plants.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host plants containing the transformed nucleic acidfragments are referred to as “transgenic” plants.

As used herein, the term “progeny,” in the context of event DP-915635-4,denotes an offspring of any generation of a parent plant which comprisescorn event DP-915635-4.

Isolated polynucleotides disclosed herein may be incorporated intorecombinant constructs, typically DNA constructs, which are capable ofintroduction into and replication in a host cell. Such a construct maybe a vector that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. A number of vectors suitable for stabletransfection of plant cells or for the establishment of transgenicplants have been described in, e.g., Pouwels et al., (1985; Supp. 1987)Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach (1989)Methods for Plant Molecular Biology, (Academic Press, New York); andFlevin et al., (1990) Plant Molecular Biology Manual, (Kluwer AcademicPublishers). Typically, plant expression vectors include, for example,one or more cloned plant genes under the transcriptional control of 5′and 3′ regulatory sequences and a dominant selectable marker. Such plantexpression vectors also can contain a promoter regulatory region (e.g.,a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

During the process of introducing an insert into the genome of plantcells, it is not uncommon for some deletions or other alterations of theinsert and/or genomic flanking sequences to occur. Thus, the relevantsegment of the plasmid sequence provided herein might comprise someminor variations. The same is possible for the flanking sequencesprovided herein. Thus, a plant comprising a polynucleotide having somerange of identity with the subject flanking and/or insert sequences iswithin the scope of the subject disclosure. Identity to the sequence ofthe present disclosure may be a polynucleotide sequence having at least65% sequence identity, at least 70% sequence identity, at least 75%sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identitywith a sequence exemplified or described herein. Hybridization andhybridization conditions as provided herein can also be used to definesuch plants and polynucleotide sequences of the subject disclosure. Asequence comprising the flanking sequences plus the full insert sequencecan be confirmed with reference to the deposited seed.

In some embodiments, two different transgenic plants can also be crossedto produce offspring that contain two independently segregating added,exogenous genes. Selfing of appropriate progeny can produce plants thatare homozygous for both added, exogenous genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation.

A “probe” is an isolated nucleic acid to which is attached aconventional, synthetic detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, or enzyme. Such aprobe is complementary to a strand of a target nucleic acid, forexample, to a strand of isolated DNA from corn event DP-915635-4 whetherfrom a corn plant or from a sample that includes DNA from the event.Probes may include not only deoxyribonucleic or ribonucleic acids butalso polyamides and other modified nucleotides that bind specifically toa target DNA sequence and can be used to detect the presence of thattarget DNA sequence.

“Primers” are isolated nucleic acids that anneal to a complementarytarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, then extended along the target DNAstrand by a polymerase, e.g., a DNA polymerase. Primer pairs refer totheir use for amplification of a target nucleic acid sequence, e.g., byPCR or other conventional nucleic-acid amplification methods. “PCR” or“polymerase chain reaction” is a technique used for the amplification ofspecific DNA segments (see, U.S. Pat. Nos. 4,683,195 and 4,800,159;herein incorporated by reference).

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence specifically in the hybridization conditions orreaction conditions determined by the operator. This length may be ofany length that is of sufficient length to be useful in a detectionmethod of choice. Generally, 11 nucleotides or more in length, 18nucleotides or more, and 22 nucleotides or more, are used. Such probesand primers hybridize specifically to a target sequence under highstringency hybridization conditions. Probes and primers according toembodiments may have complete DNA sequence similarity of contiguousnucleotides with the target sequence, although probes differing from thetarget DNA sequence and that retain the ability to hybridize to targetDNA sequences may be designed by conventional methods. Probes can beused as primers, but are generally designed to bind to the target DNA orRNA and are not used in an amplification process.

Specific primers may be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” foridentifying event DP-915635-4 in biological samples. When the probe ishybridized with the nucleic acids of a biological sample underconditions which allow for the binding of the probe to the sample, thisbinding can be detected and thus allow for an indication of the presenceof event DP-915635-4 in the biological sample. In an embodiment of thedisclosure, the specific probe is a sequence which, under appropriateconditions, hybridizes specifically to a region within the 5′ or 3′flanking region of the event and also comprises a part of the foreignDNA contiguous therewith. The specific probe may comprise a sequence ofat least 80%, from 80 and 85%, from 85 and 90%, from 90 and 95%, andfrom 95 and 100% identical (or complementary) to a specific region ofthe event.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”); Ausubel et al.eds., Current Protocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York, 1995 (with periodic updates) (hereinafter,“Ausubel et al., 1995”); and Innis et al., PCR Protocols: A Guide toMethods and Applications, Academic Press: San Diego, 1990. PCR primerpairs can be derived from a known sequence, for example, by usingcomputer programs intended for that purpose such as the PCR primeranalysis tool in Vector NTI version 6 (Informax Inc., Bethesda Md.);PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5®,1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).Additionally, the sequence can be visually scanned and primers manuallyidentified using guidelines known to one of skill in the art.

A “kit” as used herein refers to a set of reagents, and optionallyinstructions, for the purpose of performing method embodiments of thedisclosure, more particularly, the identification of event DP-915635-4in biological samples. A kit may be used, and its components can bespecifically adjusted, for purposes of quality control (e.g. purity ofseed lots), detection of event DP-915635-4 in plant material, ormaterial comprising or derived from plant material, such as but notlimited to food or feed products. “Plant material” as used herein refersto material which is obtained or derived from a plant.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences. The nucleic acid probes and primers hybridizeunder stringent conditions to a target DNA sequence. Any conventionalnucleic acid hybridization or amplification method may be used toidentify the presence of DNA from a transgenic event in a sample.

A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if they exhibit complete complementarity orminimal complementarity. As used herein, molecules are said to exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are said tobe “minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are said to be “complementary” if they can hybridize to oneanother with sufficient stability to permit them to remain annealed toone another under conventional “high-stringency” conditions.Conventional stringency conditions are described by Sambrook et al.,1989, and by Haymes et al., In: Nucleic Acid Hybridization, a PracticalApproach, IRL Press, Washington, D.C. (1985), departures from completecomplementarity are therefore permissible, as long as such departures donot completely preclude the capacity of the molecules to form adouble-stranded structure. In order for a nucleic acid molecule to serveas a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. The thermal melting point(T_(m)) is the temperature (under defined ionic strength and pH) atwhich 50% of a complementary target sequence hybridizes to a perfectlymatched probe. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the T_(m) for the specific sequence and its complement at adefined ionic strength and pH. However, in some embodiments, otherstringency conditions can be applied, including severely stringentconditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C.lower than the T_(m); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the T_(m);low stringency conditions can utilize a hybridization and/or wash at 11,12, 13, 14, 15, or 20° C. lower than the T_(m).

Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), auser may choose to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) and Sambrook et al. (1989).

In some embodiments, a complementary sequence has the same length as thenucleic acid molecule to which it hybridizes. In some embodiments, thecomplementary sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotideslonger or shorter than the nucleic acid molecule to which it hybridizes.In some embodiments, the complementary sequence is 1%, 2%, 3%, 4%, or 5%longer or shorter than the nucleic acid molecule to which it hybridizes.In some embodiments, a complementary sequence is complementary on anucleotide-for-nucleotide basis, meaning that there are no mismatchednucleotides (each A pairs with a T and each G pairs with a C). In someembodiments, a complementary sequence comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or less mismatches. In some embodiments, the complementarysequence comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or lessmismatches.

“Percent (%) sequence identity” with respect to a reference sequence(subject) is determined as the percentage of amino acid residues ornucleotides in a candidate sequence (query) that are identical with therespective amino acid residues or nucleotides in the reference sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyamino acid conservative substitutions as part of the sequence identity.Alignment for purposes of determining percent sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences (e.g., percentidentity of query sequence=number of identical positions between queryand subject sequences/total number of positions of query sequence×100).

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, stringent conditionspermit the primer pair to hybridize only to the target nucleic-acidsequence to which a primer having the corresponding wild-type sequence(or its complement) would bind and optionally to produce a uniqueamplification product, the amplicon, in a DNA thermal amplificationreaction.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether acorn plant resulting from a sexual cross contains transgenic eventgenomic DNA from the corn plant disclosed herein, DNA extracted from atissue sample of a corn plant may be subjected to a nucleic acidamplification method using a DNA primer pair that includes a firstprimer derived from flanking sequence adjacent to the insertion site ofinserted heterologous DNA, and a second primer derived from the insertedheterologous DNA to produce an amplicon that is diagnostic for thepresence of the event DNA. Alternatively, the second primer may bederived from the flanking sequence. The amplicon is of a length and hasa sequence that is also diagnostic for the event. The amplicon may rangein length from the combined length of the primer pairs plus onenucleotide base pair to any length of amplicon producible by a DNAamplification protocol. Alternatively, primer pairs can be derived fromflanking sequence on both sides of the inserted DNA so as to produce anamplicon that includes the entire insert nucleotide sequence of thePHP74643 expression construct as well as a portion of the sequenceflanking the transgenic insert. A member of a primer pair derived fromthe flanking sequence may be located a distance from the inserted DNAsequence, this distance can range from one nucleotide base pair up tothe limits of the amplification reaction. The use of the term “amplicon”specifically excludes primer dimers that may be formed in the DNAthermal amplification reaction.

Nucleic acid amplification can be accomplished by any of the variousnucleic acid amplification methods known in the art, including PCR. Avariety of amplification methods are known in the art and are described,inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in Innis etal., (1990) supra. PCR amplification methods have been developed toamplify up to 22 Kb of genomic DNA and up to 42 Kb of bacteriophage DNA(Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). Thesemethods as well as other methods known in the art of DNA amplificationmay be used in the practice of the embodiments of the presentdisclosure. It is understood that a number of parameters in a specificPCR protocol may need to be adjusted to specific laboratory conditionsand may be slightly modified and yet allow for the collection of similarresults. These adjustments will be apparent to a person skilled in theart.

The amplicon produced by these methods may be detected by a plurality oftechniques, including, but not limited to, Genetic Bit Analysis(Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNAoligonucleotide is designed which overlaps both the adjacent flankingDNA sequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (for example, using one primer in the inserted sequence andone in the adjacent flanking sequence) a single-stranded PCR product canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another detection method is the pyrosequencing technique as described byWinge (2000) Innov. Pharma. Tech. 00:18-24. In this method anoligonucleotide is designed that overlaps the adjacent DNA and insertDNA junction. The oligonucleotide is hybridized to a single-stranded PCRproduct from the region of interest (for example, one primer in theinserted sequence and one in the flanking sequence) and incubated in thepresence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. dNTPs are added individuallyand the incorporation results in a light signal which is measured. Alight signal indicates the presence of the transgene insert/flankingsequence due to successful amplification, hybridization, and single ormulti-base extension.

Fluorescence polarization as described by Chen et al., (1999) GenomeRes. 9:492-498 is also a method that can be used to detect an amplicon.Using this method an oligonucleotide is designed which overlaps theflanking and inserted DNA junction. The oligonucleotide is hybridized toa single-stranded PCR product from the region of interest (for example,one primer in the inserted DNA and one in the flanking DNA sequence) andincubated in the presence of a DNA polymerase and a fluorescent-labeledddNTP. Single base extension results in incorporation of the ddNTP.Incorporation can be measured as a change in polarization using afluorometer. A change in polarization indicates the presence of thetransgene insert/flanking sequence due to successful amplification,hybridization, and single base extension.

Quantitative PCR (qPCR) is described as a method of detecting andquantifying the presence of a DNA sequence and is fully understood inthe instructions provided by commercially available manufacturers.Briefly, in one such qPCR method, a FRET oligonucleotide probe isdesigned which overlaps the flanking and insert DNA junction. The FRETprobe and PCR primers (one primer in the insert DNA sequence and one inthe flanking genomic sequence) are cycled in the presence of athermostable polymerase and dNTPs. Hybridization of the FRET proberesults in cleavage and release of the fluorescent moiety away from thequenching moiety on the FRET probe. A fluorescent signal indicates thepresence of the flanking/transgene insert sequence due to successfulamplification and hybridization.

Molecular beacons have been described for use in sequence detection asdescribed in Tyangi et al. (1996) Nature Biotech. 14:303-308. Briefly, aFRET oligonucleotide probe is designed that overlaps the flanking andinsert DNA junction. The unique structure of the FRET probe results init containing secondary structure that keeps the fluorescent andquenching moieties in close proximity. The FRET probe and PCR primers(for example, one primer in the insert DNA sequence and one in theflanking sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae, and Curculionidaeincluding, but not limited to: Anthonomus grandis Boheman (boll weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepesabbreviatus Linnaeus (Diaprepes root weevil); Hypera punctata Fabricius(clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice waterweevil); Metamasius hemipterus hemipterus Linnaeus (West Indian caneweevil); M hemipterus sericeus Olivier (silky cane weevil); Sitophilusgranarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil);Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidusLeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden(maize billbug); S. livis Vaurie (sugarcane weevil); Rhabdoscelusobscurus Boisduval (New Guinea sugarcane weevil); flea beetles, cucumberbeetles, rootworms, leaf beetles, potato beetles, and leafminers in thefamily Chrysomelidae including, but not limited to: Chaetocnema ectypaHorn (desert corn flea beetle); C. pulicaria Melsheimer (corn fleabeetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberiSmith & Lawrence (northern corn rootworm); D. undecimpunctata howardiBarber (southern corn rootworm); D. virgifera virgifera LeConte (westerncorn rootworm); Leptinotarsa decemlineata Say (Colorado potato beetle);Oulema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferaeGoeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflowerbeetle); beetles from the family Coccinellidae including, but notlimited to: Epilachna varivestis Mulsant (Mexican bean beetle); chafersand other beetles from the family Scarabaeidae including, but notlimited to: Antitrogus parvulus Britton (Childers cane grub);Cyclocephala borealis Arrow (northern masked chafer, white grub); C.immaculata Olivier (southern masked chafer, white grub); Dermolepidaalbohirtum Waterhouse (Greyback cane beetle); Euetheola humilis rugicepsLeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French's canegrub); Tomarus gibbosus De Geer (carrot beetle); T subtropicus Blatchley(sugarcane grub); Phyllophaga crinita Burmeister (white grub); P.latifrons LeConte (June beetle); Popillia japonica Newman (Japanesebeetle); Rhzotrogus majalis Razoumowsky (European chafer); carpetbeetles from the family Dermestidae; wireworms from the familyElateridae, Eleodes spp., Melanotus spp. including M. communis Gyllenhal(wireworm); Conoderus spp.; Limonus spp.; Agriotes spp.; Ctenicera spp.;Aeolus spp.; bark beetles from the family Scolytidae; beetles from thefamily Tenebrionidae; beetles from the family Cerambycidae such as, butnot limited to, Migdolus fryanus Westwood (longhorn beetle); and beetlesfrom the Buprestidae family including, but not limited to, Aphanisticuscochinchinae seminulum Obenberger (leaf-mining buprestid beetle).

In some embodiments the DP-915635-4 maize event may further comprise astack of additional traits. Plants comprising stacks of polynucleotidesequences can be obtained by either or both of traditional breedingmethods or through genetic engineering methods. These methods include,but are not limited to, breeding individual lines each comprising apolynucleotide of interest, transforming a transgenic plant comprising agene disclosed herein with a subsequent gene and co- transformation ofgenes into a single plant cell. As used herein, the term “stacked”includes having the multiple traits present in the same plant (i.e.,both traits are incorporated into the nuclear genome, one trait isincorporated into the nuclear genome and one trait is incorporated intothe genome of a plastid or both traits are incorporated into the genomeof a plastid).

In some embodiments the DP-915635-4 maize event disclosed herein, aloneor stacked with one or more additional insect resistance traits can bestacked with one or more additional input traits (e.g., herbicideresistance, fungal resistance, virus resistance, stress tolerance,disease resistance, male sterility, stalk strength, and the like) oroutput traits (e.g., increased yield, modified starches, improved oilprofile, balanced amino acids, high lysine or methionine, increaseddigestibility, improved fiber quality, drought resistance, and thelike). Thus, the embodiments can be used to provide a complete agronomicpackage of improved crop quality with the ability to flexibly and costeffectively control any number of agronomic pests.

In a further embodiment, the DP-915635-4 maize event may be stacked withone or more additional Bt insecticidal toxins, including, but notlimited to, a Cry3B toxin disclosed in U.S. Pat. Nos. 8,101,826,6,551,962, 6,586,365, 6,593,273, and PCT Publication WO 2000/011185; amCry3B toxin disclosed in U.S. Pat. Nos. 8,269,069, and 8,513,492; amCry3A toxin disclosed in U.S. Pat. Nos. 8,269,069, 7,276,583 and8,759,620; or a Cry34/35 toxin disclosed in U.S. Pat. Nos. 7,309,785,7,524,810, 7,985,893, 7,939,651 and 6,548,291. In a further embodiment,the DP-915635-4 maize event may be stacked with one or more additionaltransgenic events containing these Bt insecticidal toxins and otherColeopteran active Bt insecticidal traits for example, event MON863disclosed in U.S. Pat. No. 7,705,216; event MIR604 disclosed in U.S.Pat. No. 8,884,102; event 5307 disclosed in U.S. Pat. No. 9,133,474;event DAS-59122 disclosed in U.S. Pat. No. 7,875,429; event DP-4114disclosed in U.S. Pat. No. 8,575,434; event MON 87411 disclosed in U.S.Pat. No. 9,441,240; event DP-23211 disclosed in International PatentApplication Publication Number WO 2019/209700; and event MON88017disclosed in U.S. Pat. No. 8,686,230 all of which are incorporatedherein by reference. In some embodiments, the DP-915635-4 maize eventmay be stacked with MON-87429-9 (MON87429 Event); MON87403; MON95379;MON87427; MON87419; MON-00603-6 (NK603); MON-87460-4; LY038;DAS-06275-8; BT176; BT11; MIR162; GA21; MZDTO9Y; SYN-05307-1; andDAS-40278-9.

In some embodiments, the disclosed compositions can be introduced intothe genome of a plant using genome editing technologies, or previouslyintroduced polynucleotides in the genome of a plant may be edited usinggenome editing technologies. For example, the disclosed polynucleotidescan be introduced into a desired location in the genome of a plantthrough the use of double-stranded break technologies such as TALENs,meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Forexample, the disclosed polynucleotides can be introduced into a desiredlocation in a genome using a CRISPR-Cas system, for the purpose ofsite-specific insertion. The desired location in a plant genome can beany desired target site for insertion, such as a genomic region amenablefor breeding or may be a target site located in a genomic window with anexisting trait of interest. Existing traits of interest could be eitheran endogenous trait or a previously introduced trait.

In some embodiments, where the disclosed polynucleotide has previouslybeen introduced into a genome, genome editing technologies may be usedto alter or modify the introduced polynucleotide sequence. Site specificmodifications that can be introduced into the disclosed compositionsinclude those produced using any method for introducing site specificmodification, including, but not limited to, through the use of generepair oligonucleotides (e.g. US Publication 2013/0019349), or throughthe use of double-stranded break technologies such as TALENs,meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Suchtechnologies can be used to modify the previously introducedpolynucleotide through the insertion, deletion or substitution ofnucleotides within the introduced polynucleotide. Alternatively,double-stranded break technologies can be used to add additionalnucleotide sequences to the introduced polynucleotide. Additionalsequences that may be added include, additional expression elements,such as enhancer and promoter sequences. In another embodiment, genomeediting technologies may be used to position additionalinsecticidally-active proteins in close proximity to the disclosedcompositions disclosed herein within the genome of a plant, in order togenerate molecular stacks of insecticidally-active proteins.

An “altered target site,” “altered target sequence.” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

In some embodiments, a corn plant comprising a DP-915635-4 event may betreated with a seed treatment. In some embodiments, the seed treatmentmay be a fungicide, an insecticide, or a herbicide.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1. Cassette Design for Transgenic Plants ContainingConstructs Encoding IPD079Ea

Cassette designs for IPD079Ea expression used in the molecular stacks togenerate commercial track events was chosen based upon efficacy andexpression in gene testing transformation experiments. A large number ofdifferent regulatory (promoters, introns) and other elements(terminators) were evaluated in gene testing experiments. The largenumber of different regulatory elements were used to evaluate expressionpatterns for yield and trait efficacy.

The genetic elements contained in the IPD079Ea gene cassette of T-DNARegion of the selected event construct, Plasmid PHP83175, are describedin Table 1.

TABLE 1 Description of Genetic Elements in the T-DNA Region of PlasmidPHP83175 Location on T-DNA (Base Size Pair Position) Genetic Element(bp) Description 20,120-20,157 Intervening Sequence 38 DNA sequence usedfor cloning ipd079Ea 20,158-21,738 sb-RCc3 Enhancer 1581 Enhancerregion, showing root-specific gene activity, from the Sorghum bicolor(sorghum) cassette root cortical RCc3 (sb-RCc3) gene 21,739-21,744Intervening Sequence 6 DNA sequence used for cloning 21,745-23,325sb-RCc3 Enhancer 1581 Enhancer region, showing root-specific activity,from the Sorghum bicolor (sorghum) root cortical RCc3 (sb-RCc3) gene (WOPatent 2012112411) 23,326-23,338 Intervening Sequence 13 DNA sequenceused for cloning 23,339-24,922 sb-RCc3 Enhancer 1584 Enhancer region,showing root-specific activity, from the Sorghum bicolor (sorghum) rootcortical RCc3 (sb-RCc3) gene (WO Patent 2012112411) 24,923-25,833zm-PCOa Promoter 911 Promoter region upstream of a Zea mays PCO118362mRNA sequence identified as having root-specific activity (WO Patent2017222821) 25,834-25,851 Intervening Sequence 18 DNA sequence used forcloning 25,852-26,707 zm-HPLV9 Intron 856 Intron region from the Zeamays ortholog of an Oryza sativa (rice) hypothetical protein (zm- HPLV9)gene, a predicted Zea mays calmodulin 5 gene (Phytozome gene IDZm00008a029682, WO Patent 2016109157) 26,708-26,716 Intervening Sequence9 DNA sequence used for cloning 26,717-28,156 ipd079Ea 1440 Insecticidalprotein gene from Ophioglossum pendulum (WO Patent 2017023486)28,157-28,173 Intervening Sequence 17 DNA sequence used for cloning28,174-29,126 sb-SCI-1B Terminator 953 Terminator region of the Sorghumbicolor (sorghum) subtilisin-chymotrypsin inhibitor 1B gene (WO Patent2018102131) 29,127-29,172 Intervening Sequence 46 DNA sequence used forcloning 29,173-29,632 Z27G Terminator 460 Terminator region from the Zeamays W64 line 27-kDa gamma zein gene (Das et al., 1991; Liu et al.,2016)

Example 2. Transformation of Maize by Agrobacterium Transformation andRegeneration of Transgenic Plants Containing the IPD079, PAT, and PMIGenes

DP-915635-4 maize event was produced by Agrobacterium-mediated SSItransformation with plasmid PHP83175. Agrobacterium-mediated SSI wasessentially performed as described in U.S. patent applicationpublication number 2017/0240911, herein incorporated by reference.

Over 2700 immature embryos were infected with PHP83175. After the105-day selection and regeneration process, a total of 46 T0 plantletswere regenerated. Samples were taken from all T0 plantlets for PCRanalysis to verify the presence and copy number of the inserted IPD079,PMI, and mo-PAT. In addition to this analysis, the T0 plantlets wereanalyzed by PCR for the presence of certain Agrobacterium binary vectorbackbone sequences and for the developmental genes, zm-odp2 and zm-wus2disclosed in U.S. Pat. Nos 7,579,529 and 7,256,322, herein incorporatedby reference in their entireties. Plants that were determined to containsingle copy of the inserted genes, no Agrobacterium backbone sequences,and no developmental genes were selected for further greenhousepropagation. Samples from those PCR selected T0 quality events werecollected for further analysis using Southern-by-Sequencing to confirmthat the inserted genes were in the correct target locus without anygene disruptions. Maize events DP-915635-4 were confirmed to contain asingle copy of the T-DNA (See Examples 3 and 4). These selected T0plants were assayed for trait efficacy and protein expression. T0 plantsmeeting all criteria were advanced and crossed to inbred lines toproduce seed for further testing. A schematic overview of thetransformation and event development is presented in FIG. 4 .

Example 3. Identification of Maize Events DP-915635-4

Genomic DNA from leaf tissue representing multiple generations of maizeevent DP-915635-4, known copy number calibrator controls, a negativecontrol source (DNA from a non-genetically modified maize) and notemplate controls (NTC) were isolated and subjected to quantitativereal-time PCR (qPCR) amplification using event-specific andconstruct-specific primer and probes. Real-time PCR analyses ofDP-915635-4 maize DNA using event-specific and construct-specific assaysconfirm the stable integration and segregation of a single copy of theT-DNA of plasmid PHP83175 in leaf samples tested, as demonstrated by thequantified detection of event DP-915635-4, and ipd079Ea, pmi, and mo-PATtransgenes in DP-915635-4 maize. The reliability of each event-specificand construct-specific PCR method was assessed by repeating theexperiment in quadruplicate. The sensitivity, or Limit of Detection(LOD) of the PCR amplification was evaluated by testing of variousdilutions of the genomic DNA from DP-915635-4.

Two generations of maize containing event DP-915635-4 were grown incell-divided flats under typical greenhouse production conditions.Approximately 100 seed were planted for each generation.

Leaf samples were collected from each healthy plant, when plants werebetween the V5 and V9 growth stages. The samples were taken from theyoungest leaf that was emerged from the whorl of each plant. Three leafpunches per plant were analyzed for the copy number of each event'sgenomic junction and the PHP83175 T-DNA through copy number PCR (qPCR)for the DP-915635-4event as well as ipd079Ea, pmi, and mo-PAT transgenesfrom seed grown at Pioneer Hi-Bred International, Inc. (Johnston, Iowa).Genomic DNA extractions from the leaf samples were performed using ahigh alkaline extraction protocol. Validated laboratory controls (copynumber calibrators and negative) were prepared from leaf tissue using astandard cetyl trimethylammonium bromide (CTAB) extraction protocol.

Genomic DNA supporting laboratory controls were quantified usingQuant-iT PicoGreen® reagent (Invitrogen, Carlsbad, Calif.).Quantification of genomic test and control samples were estimated usingthe NanoDrop 2000c Spectrophotometer using NanoDrop 2000/2000c V1.6.198Software (ThermoScientific, Wilmington, Del.).

Genomic DNA samples isolated from leaf tissue of DP-915635-4 as well ascontrol samples were subjected to real-time PCR amplification utilizingevent-specific and construct specific primers and probes which spanspecific regions of the PHP83175 T-DNA as well as the genomic junctionsthat span each insertion site for events DP-915635-4. An endogenousreference gene, High Mobility Group A (hmg-A) (Krech, et al. (1999).Gene 234: (1) 45-50) was used in duplex with each assay for bothqualitative and quantitative assessment of each assay and to demonstratethe presence of sufficient quality and quantity of DNA within the PCRreaction. The PCR target sites and size of expected PCR products foreach primer/probe set are shown in Table 2. Primer and probe sequenceinformation supporting each targeted region are shown in Table 3. PCRreagents and reaction conditions are shown in Table 4. In this studyapproximately 3-ng of maize genomic DNA was used for all PCR reactions.

TABLE 2 PCR Genomic DNA Target Site and Expected Size of PCR ProductsTargeted Expected Size of Amplicon Primer and Probe Set Regions PCRProduct (bp) SEQ ID NO: SEQ ID NOs: 6-8 DP-915635-4 72 21 insertion SEQID NOs: 9-11 ipd079Ea 57 22 SEQ ID NOs: 12-14 pmi 113 23 SEQ ID NOs:15-17 mo-PAT 76 24 SEQ ID NOs: 18-20 hmg-A 79 25

TABLE 3 Primers and Probe Sequence and Amplicon for PCR Genomic DNATargeted Regions Reagent Sequence (5’ to 3’) Length (base) SEQ ID NO: 6GCATCTAGGACCGACTAGCTAACTAAC 27 forward primer SEQ ID NO: 7CTTTGCATCATGTCTTGAACAATG 24 reverse primer SEQ ID NO: 86-FAM-CGCCATGAGGAGCAA-MGB 15 probe SEQ ID NO: 9 GCTGGCCGTGAAGGTGAA 18forward primer SEQ ID NO: 10 TCCACGCTAGCGCTGAAGTA 20 reverse primerSEQ ID NO: 11 6-FAM-CTCAGCGGAAGCTA-MGB 14 probe SEQ ID NO: 12TGACTGTCAAAGGCCACGG 19 forward primer SEQ ID NO: 13AGATGGACAAGTCTAGGTTCCACC 24 reverse primer SEQ ID NO: 14 6-FAM- 28 probeCCGTTTAGCGCGTGTTTACAACAAGCTG-BHQ SEQ ID NO: 15 CATCGTGAACCACTACATCGAGAC24 forward primer SEQ ID NO: 16 GTCGATCCACTCCTGCGG 18 reverse primerSEQ ID NO: 17 6'FAM-ACCGTGAACTTCCGCACCGAGC-BHQ1 22 probe SEQ ID NO: 18TTGGACTAGAAATCTCGTGCTGA 23 forward primer SEQ ID NO: 19GCTACATAGGGAGCCTTGTCCT 22 reverse primer SEQ ID NO: 20VIC-GCGTTTGTGTGGATTG-MGB 16 probeSEQ ID NO: 21: DP-915635-4 assay amplicon sequence (72-bp; primer and probe bindingsites are in bold and underlined) GCATCTAGGACCGACTAGCTAACTAAC TAGGGCGCCATGAGGAGCAA T CATTGTTCAAGACATGATGCAAAGSEQ ID NO: 22: ipd079Ea assay amplicon sequence (57-bp; primer and probe bindingsites are in bold and underlined) GCTGGCCGTGAAGGTGAA G CTCAGCGGAAGCTACGGC TACTTCAGCGC TAGCGTGGASEQ ID NO: 23: pmi assay amplicon sequence (113-bp; primer and probe binding sitesare in bold and underlinedTGACTGTCAAAGGCCACGGCCGTTTAGCGCGTGTTTACAACAAGCTG TAAGAGCTTACTGAAAAAATTAACATCTCTTGCTAAGCTGGG GGTGGAACCTAGACTTGTCCA TCTSEQ ID NO: 24: mo-pat assay amplicon sequence (76-bp; primer and probe binding sitesare in bold and underlined CATCGTGAACCACTACATCGAGAC CTCCACCGTGAACTTCCGCACCGAGC CGCA GACC CCGCAGGAGTGGATCGACSEQ ID NO: 25: hmg-A assay amplicon sequence (79-bp; primer and probe binding sitesare in bold and underlined) TTGGACTAGAAATCTCGTGCTGA TTAATTGTTTTACGCGTGCGTTTGTGTGGATT G T AGGACAAGGCTCCCTATGTAGC

TABLE 4 PCR Reagents and Reaction Conditions Temperature Time StepDescription (° C.) (seconds) Cycles 1 Initial Denaturation 95 120 1 2aAmplification Denaturation 95 1 40^(a ) 2b Anneal/ 60 20 Extend ^(a)Ifthermal cycling is completed using a Roche LightCycler ® 480, initialdenaturation was 300 seconds for step 1 with 45 cycles for steps 2a and2b.

PCR products ranging in size from 57-bp to 113-bp, representing theinsertion sites for event DP-915635-4 as well as the transgenes withinthe T-DNA from plasmid PHP83175, were amplified and observed in 100individual leaf samples from event DP-915635-4 as well as eight copynumber calibrator genomic controls, but were absent in each of the eightnegative genomic controls and eight NTC controls. Each assay wasperformed a total of four times with the same results observed. C_(T)values were calculated for each sample and all positive controls.

Using the maize endogenous reference gene hmg-A, a PCR product of 79-bpwas amplified and observed in 100 individual leaf samples each fromevent DP-915635-4 as well as eight copy number calibrator and eightnegative genomic controls. Amplification of the endogenous gene was notobserved in the eight No Template (NTC) controls tested with nogeneration of C_(T) values. For each sample, each assay was performed induplex with both insertion sites and all transgenes a total of fourtimes with the same results observed each time. C_(T) values werecalculated for each sample and all positive and negative controls.

To assess the sensitivity of the construct-specific PCR assays,DP-915635-4 maize DNA was diluted in control maize genomic DNA,resulting in test samples containing various amounts of eventDP-915635-4 DNA (5-ng, 1-ng, 500-pg, 250pg, 100-pg, 50-pg, 20-pg, 10-pg,5-pg) in a total of 5-ng maize DNA. These various amounts of DP-915635-4maize DNA correspond to 100%, 20%, 10%, 5%, 2%, 1%, 0.4%, 0.2%, and 0.1%of DP-915635-4 maize maize DNA in total maize genomic DNA, respectively.The various amounts of DP-915635-4 DNA were subjected to real-time PCRamplification for transgenes ip079Ea, PMI, and mo-PAT Based on theseanalyses, the limit of detection (LOD) in 5-ng of total DNA for eventDP-915635-4 was determined to be approximately 20-pg for ip079Ea, or0.4%, 250-pg for pmi, or 5%, and 5-pg for mo-pat, or 0.1% (DP-915635-4).The determined sensitivity of each assay described is sufficient formany screening applications. Each concentration was tested a total offour times with the same results observed each time.

Real-time PCR analyses of event DP-915635-4 utilizing event-specific andconstruct-specific primer/probe sets for event DP-915635-4 confirm thestable integration and segregation of a single copy of the T-DNA ofplasmid PHP83175 of the event in leaf samples tested, as demonstrated bythe quantified detection of ipd079Ea, pmi, and mo-PAT transgenes inDP-915635-4 maize. These results were reproducible among all thereplicate qPCR analyses conducted. The maize endogenous reference geneassay for detection of hmg-A amplified as expected in all the testsamples, negative controls and was not detected in the NTC samples. Thesensitivity of each assay under the conditions described ranges from5-pg to 250-pg DNA, all sufficient for many screening applications byPCR.

Example 4. Southern-by-Sequencing (SbS) Analysis of DP-915635-4 maizefor Integrity and Copy Number

Southern-by-Sequencing (SbS) utilizes probe-based sequence capture, NextGeneration Sequencing (NGS) techniques, and bioinformatics procedures toisolate, sequence, and identify inserted DNA within the maize genome. Bycompiling a large number of unique sequencing reads and comparing themto the transformation plasmid, unique junctions due to inserted DNA areidentified in the bioinformatics analysis and can be used to determinethe number of insertions within the plant genome. The T0 plant ofDP-915635-4 maize was analyzed by SbS to determine the insertion copynumber. In addition, samples of the control maize line were analyzed.

Genomic DNA was extracted from the T0 generation of DP-915635-4 maizeand control plants.

Capture probes used to select PHP83175 plasmid sequences were designedand synthesized by Roche NimbleGen, Inc. (Madison, Wis.). A series ofunique sequences encompassing the plasmid sequence was used to designoverlapping biotinylated oligonucleotides as capture probes. The probeset was designed to target most sequences within the PHP83175transformation plasmid during the enrichment process. The probes werecompared to the maize genome to determine the level of maize genomicsequence that would be captured and sequenced simultaneously with thePHP83175 plasmid sequence.

Next-generation sequencing libraries were constructed for theDP-915635-4 maize plants and the control maize lines. SbS was performedas described by Zastrow-Hayes, et al. Plant Genome (2015). Thesequencing libraries were hybridized to the capture probes through tworounds of hybridization to enrich the targeted sequences. Following NGSon a HiSeq 2500 (Illumina, San Diego, Calif.), the sequencing reads wereassessed for trimming and quality assurance. Reads were aligned againstthe maize genome and the transformation construct and reads that containboth genomic and plasmid sequence were identified as junction reads.Alignment of the junction reads to the transformation construct showsborders of the inserted DNA relative to the expected insertion.

To identify junctions that included endogenous maize sequences, controlmaize genomic DNA libraries were captured and sequenced in the samemanner as the DP-915635-4 maize plants. These libraries were sequencedto an average depth approximately five times that of the depth for theDP-915635-4 maize plant samples. This increased the probability that theendogenous junctions captured by the PHP83175 probes would be detectedin the control samples, so that they could be identified and removed inthe DP-915635-4 maize samples.

Integration and copy number of the insertion were determined inDP-915635-4 maize derived from construct PHP83175. Schematic maps of thePHP83175 plasmid and the T-DNA from PHP83175 used in transformation areprovided in FIGS. 1 and 2 .

SbS was conducted on the T0 plant of DP-915635-4 maize to determine theinsertion copy number in the genome. Alignment of the SbS reads to theexpected insertion region (including the landing pad elements zm-SEQ158and zm-SEQ159; FIG. 3 ) resulted in two unique junctions between thegenomic flanking sequence and the landing pad. The FRT1 and FRT6 sitesare the two locations where the target trait genes from PHP83175 wereintegrated into the site-specific integration (SSI) landing pad. Therewere no other junctions between the PHP83175 sequences and the maizegenome detected in the plant, indicating that there are no additionalplasmid-derived insertions present in DP-915635-4 maize. Additionally,there were no junctions between non-contiguous regions of the PHP83175T-DNA identified, indicating that there are no detectable rearrangementsor truncations in the inserted DNA. Furthermore, there were no junctionsbetween maize genome sequences and the backbone sequence of PHP83175 inthe plant analyzed, demonstrating that no plasmid backbone sequenceswere incorporated into DP-915635-4 maize.

SbS analysis of the T0 plant of DP-915635-4 maize demonstrated thatthere is a single insertion containing the desired genes from thePHP83175 T-DNA in DP-915635-4 maize and that no additional insertionsare present in the respective genomes.

Southern-by-Sequencing (SbS) analysis was conducted on the T0 plant ofDP-915635-4 maize to confirm insertion copy number. The results indicatea single PHP83175 T-DNA insertion in the plant. No junctions between thePHP83175 T-DNA sequences and the maize genome were detected in controlplants, indicating that, as expected, these plants did not contain anyinsertions derived from PHP83175. Furthermore, no plasmid backbonesequences were detected in the plant analyzed. SbS analysis of the T0plant of DP-915635-4 maize demonstrated that there is a single insertionof the PHP83175 T-DNA in DP-915635-4 maize and that no additionalinsertions are present in the respective genomes.

A single nucleotide change, A to C change at bp 2931 in the ubiZM1promoter of the complete insert and flanking region sequence as shown inSEQ ID NO: 32, was identified in all five plants in the ubiZM1 promoterof the pmi cassette that differs from the expected insertion sequence.As this change is in all five positive plants, it was determined to bepresent in the initial transformed plant. An additional singlenucleotide change of a G to an A at position 8199 in SEQ ID NO: 32 wasidentified in the os-actin promoter of one plant of five plants; as thisis the only occurrence it is likely due to a spontaneous change duringthe breeding process. Alignments of the reads from the five positiveplants to the five plasmid maps show coverage of the genetic elementsfound in the intended insertion, along with coverage of the endogenouselements in the plasmids that were not incorporated into the insertion(zm-SEQ158, zm-SEQ159, zm-U6 pol III CHR8 promoter and terminator,zm-45CR1 guide RNA, Int-2 promoter, zm-wus2, and zm-odp2). Reads alsoaligned to the pinII terminator elements located outside of the intendedinsertion regions in PHP83175, PHP73878, PHP70605, and PHP21875 althoughthese elements were not incorporated into the insertion. The NGS readsthat aligned to these copies of the pinII terminator are from fragmentscontaining the pinII terminator in the pmi cassette of the intendedinsertion; however, the reads from this single copy align to all copiesof the pinII terminator in the plasmid maps. Similarly, reads aligned tothe CaMV 35S terminator elements in the mo-Flp cassette and to theos-actin promoter and intron region of the zm-wus2 cassette in PHP83175due to the presence of identical elements in the mo-pat cassette of theintended insertion.

Example 5. Insect Efficacy of Maize Events DP-915635-4

F1 hybrid maize lines containing the insect-active IPD079Ea protein wereevaluated in the field for protection against corn rootworms (CRW) bytesting multiple events including DP-915635-4 maize. Data werestatistically analyzed using a linear mixed model.

Field testing was conducted in 13 locations located in commercialmaize-growing regions of North America: Brookings, S. Dak. (BR);Mankato, Minn. (MK); Marion, Iowa (MR); Readlyn, Iowa (MR_RE); Johnston,Iowa (JH_D2); Johnston, Iowa (JH_D3); Gilbert, Iowa (JH_GB); Watertown,Wis. (JV); Shabonna, Ill. (JV_SH); Seymour, Ill. (CI_SE); Fowler, Ind.(WN); York, Nebr. (YK); and Lindsey, Nebr. (YK_LI). No efficacy datawere collected at six of the 13 locations (sites JH_GB, JV, JV_SH,CI_SE, YK, & YK_LI) due to a low nodal injury score (CRWNIS) below 0.75on negative control roots.

Single-row plots (10 feet in length) were planted in a randomizedcomplete block experimental design with two replications. Prior toplanting, 168 kernels from each seed lot were characterized by PCRanalysis to confirm the presence of the traits. A four-foot length ofeach row was manually infested utilizing a tractor-mounted CRW egginfester at a targeted infestation rate of approximately 750 eggs/plantor 1500 eggs/plant, depending on the location, when plants reached theV2-V4 growth stages. Eggs were injected into the soil approximately 4inches deep and approximately 2-3 inches on both sides of each plant.Injury from larval feeding on roots was evaluated between 56 and 71 daysafter planting. Two corn roots were tagged, manually dug from theground, washed clean of soil with pressurized water, and evaluated forthe amount of larval feeding at approximately the R2 growth stage. Rootinjury was evaluated by visually rating and recording the amount oflarval feeding contained on each root using the Iowa State 0-3node-injury scale.

The mean node-injury root rating results from CRW for both DP-915635-4maize and control maize are provided in Table 5. These results indicatethat maize lines containing the insect-active IPD079Ea protein areefficacious against CRW.

TABLE 5 Efficacy Results Against Corn Rootworm Mean Node- Maize Numberof Injury Root Line Plots Rating ± SD Range P-Value DP915635 14 0.16 ±0.17 0.02-0.71 <0.0001^(a) Control 14 2.02 ± 0.80 0.70-3.00^(a)Statistically significant difference; (P-value < 0.05)

TABLE 6 Efficacy Results Against Corn Rootworm Mean Node- Number ofInjury Root Maize Line Plots Rating ± SD Range P-Value DP-915635-4 270.13 + 0.08 0.02-0.70 <0.0001^(a) Control 27 1.79 + 0.74 0.50-3.00^(a)Statistically significant difference; (P-value < 0.05)

Example 6. Agronomic and Yield Field Evaluations of Maize EventsDP-915635-4

Agronomic field trials containing DP-915635-4 were to generate yielddata and to evaluate other agronomic characteristics. All inbred andhybrid materials tested for an event were generated from a single T0plant.

Hybrid Trials

Hybrid trials were planted at 16 locations with a single replicate ofthe entry list at each location. Grain was harvested from 12 of the 16locations. Each entry in a common background was crossed to threetesters to generate hybrid seed for testing. Experiments were nested bytesters, with the entries randomized within each nest. Variousobservations and data were collected at each planted location throughoutthe growing season. The following agronomic characteristics wereanalyzed for comparison to a wild type entry (WT), or an entry with thesame genetics but without IPD079Ea, also referred to as base comparator(Table 7 and FIG. 5 ):

-   -   1.) Ear height (EARHT): Measurement from the ground to the        attachment point of the highest developed ear on the plant. Ear        height is measured in inches.    -   2.) Plant height (PLTHT): Measurement by drones from the ground        to the base of the flag leaf. Plant height is measured in        inches.    -   3.) Moisture (MST): Measurement of the percent grain moisture at        harvest.    -   4.) Yield: Recorded weight of grain harvested from each plot.        Calculations of reported bu/acre yields were made by adjusting        to measured moisture of each plot.

Inbred Trials

Inbred trials were planted at 8 locations with 2 replicates of the entrylist at each location. Grain was harvested from 7 locations foranalysis. One replicate at each location was nested by construct design;the other replicate was planted as a randomized complete block.Agronomic data and observations were collected for the inbred trials andanalyzed for comparison to a wild type entry (WT), or untraited versionof the same genotype. Data generated for the inbred trials included thefollowing agronomic traits (Table 8 and FIG. 6 ):

-   -   1.) Growing degree units to shed (GDUSHD): Measurement records        the total accumulated growing degree units when 50% of the        plants in the plot have tassels that are shedding pollen. A        single day equivalent is approximately 2.5 growing degrees units        for this data set.    -   2.) Ear height (EARHT): Measurement from the ground to the        attachment point of the highest developed ear on the plant. Ear        height is measured in inches.    -   3.) Plant height (PLTHT): Measurement from the ground to the        base of the flag leaf. Plant height is measured in inches.    -   4.) Ear photometry yield (PHTYLD): Calculated yield estimates        from images of harvested ears from each plot. Units for the        values shown are bu/acre.

Trial Results

To evaluate the hybrid data, a mixed model framework was used to performmulti location analysis. In the multi-location analysis, main effectconstruct design is considered as fixed effect. Factors for location,background, tester, event, background by construct design, tester byconstruct design, tester by event, location by background, location byconstruct design, location by tester, location by background byconstruct design, location by tester by construct design, location byevent, location by tester by event are considered as random effects. Thespatial effects including range and plot within locations wereconsidered as random effects to remove the extraneous spatial noise. Theheterogeneous residual was assumed with autoregressive correlation asAR1*AR1 for each location. The estimate of construct design andprediction of event for each background were generated. The T-tests wereconducted to compare construct design/event with WT. A difference wasconsidered statistically significant if the P-value of the differencewas less than 0.05. Yield analysis was by ASREML (VSN International Ltd;Best Linear Unbiased Prediction; Cullis, B. Ret al (1998) Biometrics 54:1-18, Gilmour, A. R. et al (2009); ASReml User Guide 3.0, Gilmour, A.R., et al (1995) Biometrics 51: 1440-50).

To evaluate the inbred data, a mixed model framework was used to performmulti location analysis. In the multi-location analysis, main effectconstruct design is considered as fixed effect. Factors for location,background, event, background by construct design, location bybackground, location by construct design, location by background byconstruct design, location by event and rep within location areconsidered as random effects. The spatial effects including range andplot within locations were considered as random effects to remove theextraneous spatial noise. The heterogeneous residual was assumed withautoregressive correlation as AR1*AR1 for each location. The estimate ofconstruct design and prediction of event for each background weregenerated. The T-tests were conducted to compare construct design/eventwith WT. A difference was considered statistically significant if theP-value of the difference was less than 0.05. Yield analysis was byASREML (VSN International Ltd; Best Linear Unbiased Prediction; Cullis,B. Ret al (1998) Biometrics 54: 1-18, Gilmour, A. R. et al (2009);ASReml User Guide 3.0, Gilmour, A. R., et al (1995) Biometrics 51:1440-50).

TABLE 7 Hybrid performance of events DP-915635-4 compared to baseentry-yield Number of Predicted Stan- Predicted Predicted plots withvalue dard lower upper Event yield data (bu/acre) Error 95% CL 95% CL WT(base 68 207.05 7.76 191.28 222.83 comparator) DP-915635-4 19 212.017.93 195.87 228.15

TABLE 8 Inbred performance of events DP-915635-4 compared to baseentry-yield Number of Predicted Stan- Predicted Predicted plots withvalue dard lower upper Event yield data (bu/acre) Error 95% CL 95% CLDP-915635-4 14 112.32 8.00 96.04 128.59 WT (base 55 129.66 7.61 114.18145.14 comparator)

Example 7. Protein Expression and Concentration Protein Extraction

For analysis of IPD079Ea protein concentrations, processed root tissuesub-samples were weighed at a target weight of 20 mg. For analysis ofPAT and PMI protein concentrations, processed leaf tissue sub-sampleswere weighed at a target weight of 10 mg. Samples were extracted with0.60 ml of chilled phosphate-buffered saline containing polysorbate 20(PBST). Extracted samples were centrifuged, and then supernatants wereremoved and prepared for analysis.

Determination of IPD079Ea Protein Concentration

Prior to analysis, samples were diluted as applicable in PBST. Standards(typically analyzed in triplicate wells) and diluted samples (typicallyanalyzed in duplicate wells) were incubated in a plate pre-coated withan IPD079Ea-specific antibody. Following incubation, unbound substanceswere washed from the plate and the bound IPD07Ea protein was incubatedwith a different IPD079Ea-specific antibody conjugated to the enzymehorseradish peroxidase (HRP). Unbound substances were washed from theplate. Detection of the bound IPD079Ea-antibody complex was accomplishedby the addition of substrate, which generated a colored product in thepresence of HRP. The reaction was stopped with an acid solution and theoptical density (OD) of each well was determined using a plate reader.

Determination of PAT Protein Concentration

Prior to analysis, samples were diluted as applicable in PBST. Standards(typically analyzed in triplicate wells) and diluted samples (typicallyanalyzed in duplicate wells) were co-incubated with a PAT-specificantibody conjugated to the enzyme HRP in a plate pre-coated with adifferent PAT-specific antibody. Following incubation, unboundsubstances were washed from the plate. Detection of the boundPAT-antibody complex was accomplished by the addition of substrate,which generated a colored product in the presence of HRP. The reactionwas stopped with an acid solution and the OD of each well was determinedusing a plate reader.

Determination of PMI Protein Concentration

Prior to analysis, samples were diluted as applicable in PBST. Standards(typically analyzed in triplicate wells) and diluted samples (typicallyanalyzed in duplicate wells) were incubated in a plate pre-coated with aPMI-specific antibody. Following incubation, unbound substances werewashed from the plate and the bound PMI protein was incubated with adifferent PMI-specific antibody conjugated to the enzyme HRP. Unboundsubstances were washed from the plate. Detection of the boundPMI-antibody complex was accomplished by the addition of substrate,which generated a colored product in the presence of HRP. The reactionwas stopped with an acid solution and the OD of each well was determinedusing a plate reader.

Calculations for Determining Protein Concentrations

SoftMax Pro GxP (Molecular Devices) microplate data software was used toperform the calculations required to convert the OD values obtained foreach set of sample wells to a protein concentration value.

A standard curve was included on each ELISA plate. The equation for thestandard curve was derived by the software, which used a quadratic fitto relate the OD values obtained for each set of standard wells to therespective standard concentration (ng/ml).

Adjusted Concentration=Interpolated Sample Concentration×Dilution Factor

Adjusted sample concentration values obtained from SoftMax Pro GxPsoftware were converted from ng/ml to ng/mg sample weight as follows:

$\begin{matrix}{{Sample}{Concentration}} & {Sample} & {{Extraction}{Buffer}} \\{\left( {{ng}{protein}/{mg}{sample}} \right.} & {Concen} & \frac{{Volume}({ml})}{{Sample}{Target}} \\\left. {weight} \right) & {{tration}\left( {{ng}/{ml}} \right.} & {{Weight}({mg})}\end{matrix}$

Results

Protein concentration results (means, standard deviations, and ranges)were determined for IPD079Ea protein in V9 root tissue and the PAT andPMI proteins in V9 leaf tissue from two generations of DP-915635-4maize.

TABLE 9 Expressed Trait Protein Concentration Results ProteinConcentration (ng/mg Tissue Dry Weight) Number of Samples < Tissue(Growth Sample LLOQ/Total Number of Protein Stage) Generation Mean ± SDRange LLOQ Samples Reported IPD079Ea Root (V9) T3 22 ± 7.0  14-29 0.0690/5 F1 20 ± 3.1  15-23 0.069 0/5 PAT Leaf (V9) T3 6.3 ± 0.64 5.6-7.20.11 0/5 F1 5.1 ± 0.63 4.2-5.9 0.11 0/5 PMI Leaf (V9) T3 11 ± 2.5 7.8-14  0.54 0/5 F1 5.8 ± 0.86 4.9-7.2 0.54 0/5

The above description of various illustrated embodiments of thedisclosure is not intended to be exhaustive or to limit the scope to theprecise form disclosed. While specific embodiments of and examples aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. The teachings providedherein can be applied to other purposes, other than the examplesdescribed above. Numerous modifications and variations are possible inlight of the above teachings and, therefore, are within the scope of theappended claims.

These and other changes may be made in light of the above detaileddescription. In general, in the following claims, the terms used shouldnot be construed to limit the scope to the specific embodimentsdisclosed in the specification and the claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books or otherdisclosures) in the Background, Detailed Description, and Examples isherein incorporated by reference in their entireties.

Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight, molecular weight isaverage molecular weight; temperature is in degrees celsius; andpressure is at or near atmospheric.

What is claimed is:
 1. A corn plant comprising the genotype of the cornevent DP-915635-4, wherein said genotype comprises a nucleotide sequenceas set forth in SEQ ID NO: 26 and SEQ ID NO:
 29. 2. The corn plant ofclaim 1, wherein said genotype comprises the nucleotide sequence setforth in SEQ ID NO: 27 and SEQ ID NO:
 30. 3. The corn plant of claim 1,wherein said genotype comprises the nucleotide sequence set forth in SEQID NO: 28 and SEQ ID NO:
 31. 4. A DNA construct comprising an operablylinked first and second expression cassette, wherein said firstexpression cassette comprises: 1) an sb-RCc3 Enhancer 2) a zm-PCOaPromoter; 3) a zm-HPLV9 Intron; 4) an ipd079Ea; and 5) an sb-SCI-1BTerminator.
 5. A plant comprising the DNA construct of claim
 4. 6. Theplant of claim 5, wherein said plant is a corn plant.
 7. A plantcomprising the sequence set forth in SEQ ID NO:
 21. 8. A corn eventDP-915635-4, wherein a representative sample of seed of said corn eventhas been deposited with American Type Culture Collection (ATCC) withAccession No. PTA-126746.
 9. Plant parts of the corn event of claim 8.10. Seed comprising corn event DP-915635-4, wherein said seed comprisesa DNA molecule chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein arepresentative sample of the corn event DP-915635-4 seed of has beendeposited with American Type Culture Collection (ATCC) with AccessionNo. PTA-126746.
 11. A corn plant, or part thereof, grown from the seedof claim
 10. 12. A transgenic seed produced from the corn plant of claim8.
 13. A transgenic corn plant, or part thereof, grown from the seed ofclaim
 12. 14. An isolated nucleic acid molecule comprising a nucleotidesequence chosen from SEQ ID NOs: 21, and 26-31, and full lengthcomplements thereof.
 15. (canceled)
 16. A biological sample derived fromcorn event DP-915635-4 plant, tissue, or seed, wherein said samplecomprises a nucleotide sequence which is or is complementary to asequence chosen from SEQ ID NO: 26 and SEQ ID NO: 29, wherein saidnucleotide sequence is detectable in said sample using a nucleic acidamplification or nucleic acid hybridization method, wherein arepresentative sample of said corn event DP-915635-4 seed has beendeposited with American Type Culture Collection (ATCC) with AccessionNo. PTA-126746.
 17. The biological sample of claim 16, wherein saidbiological sample comprises plant, plant tissue, or seed of transgeniccorn event DP-915635-4 .
 18. The biological sample of claim 17, whereinsaid biological sample is a DNA sample extracted from the transgeniccorn plant event DP-915635-4, and wherein said DNA sample comprises oneor more of the nucleotide sequences chosen from SEQ ID NOs: 21-31, andthe complement thereof
 19. The biological sample of claim 16, whereinsaid biological sample is chosen from corn flour, corn meal, corn syrup,corn oil, corn starch, and cereals manufactured in whole or in part tocontain corn by-products.
 20. An extract derived from corn eventDP-915635-4 plant, tissue, or seed and comprising a nucleotide sequencewhich is or is complementary to a sequence chosen from SEQ ID NO: 26 andSEQ ID NO: 29, wherein a representative sample of said corn eventDP-915635-4 seed has been deposited with American Type CultureCollection (ATCC) with Accession No. PTA-126746.
 21. The extract ofclaim 20, wherein said nucleotide sequence is detectable in said extractusing a nucleic acid amplification or nucleic acid hybridization method.22. The extract of claim 21, wherein said extract comprises plant, planttissue, or seed of transgenic corn plant event DP-915635-4.
 23. Theextract of claim 22, wherein the extract is a composition chosen fromcorn flour, corn meal, corn syrup, corn oil, corn starch, and cerealsmanufactured in whole or in part to contain corn by-products, whereinsaid composition comprises a detectable amount of said nucleotidesequence.
 24. A method of producing hybrid corn seeds comprising: A)sexually crossing a first inbred corn line comprising a nucleotidechosen from SEQ ID NOs: 21-31 and a second inbred line having adifferent genotype; B) growing progeny from said crossing; and C)harvesting the hybrid seed produced thereby.
 25. The method according toclaim 24, wherein the first inbred corn line is a female parent.
 26. Themethod according to claim 24, wherein the first inbred corn line is amale parent.
 27. A method for producing a corn plant resistant tocoleopteran pests comprising: A) sexually crossing a first parent cornplant with a second parent corn plant, wherein said first or secondparent corn plant comprises event DP-915635-4 thereby producing aplurality of first generation progeny plants; B) selfing the firstgeneration progeny plant, thereby producing a plurality of secondgeneration progeny plants; and C) selecting from the second generationprogeny plants that comprise the event DP-915635-4 and are resistant toa coleopteran pest.
 28. A method of producing hybrid corn seedscomprising: A) sexually crossing a first inbred corn line comprising theDNA construct of claim 1 with a second inbred line not comprising theDNA construct of claim 1; and B) harvesting the hybrid seed producedthereby.
 29. The method of claim 28, further comprising the step ofbackcrossing a second generation progeny plant that comprises corn eventDP-915635-4 to the parent plant that lacks the corn event DP-915635-4DNA, thereby producing a backcross progeny plant that is resistant to acoleopteran pest.
 30. A method for producing a corn plant resistant to acorn rootworm, said method comprising: A) crossing a first parent cornplant with a second parent corn plant, wherein said first or secondparent corn plant comprises event DP-915635-4, thereby producing aplurality of first generation progeny plants; B) selecting a firstgeneration progeny plant that comprises the event DP-915635-4; C)backcrossing the first generation progeny plant of step (b) with aparent plant that lacks the corn event DP-915635-4 DNA, therebyproducing a plurality of backcross progeny plants; and D) selecting fromthe backcross progeny plants, a plant that comprises the eventDP-915635-4; wherein the selected backcross progeny plant of step (d)comprises SEQ ID NO: 21, 26, or
 29. 31. The method according to claim30, wherein the plants of the first parent corn line are the femaleparents or male parents.
 32. Hybrid seed produced by the method of claim30.
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled) 37.(canceled)
 38. (canceled)
 39. (canceled)
 40. (canceled)
 41. (canceled)42. (canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled) 46.(canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled)
 50. A corn plantcomprising the genotype of the corn event DP-915635-4, wherein saidgenotype comprises a nucleotide sequence having at least 95% sequenceidentity to SEQ ID NO: 26 and SEQ ID NO:
 29. 51. The corn plant of claim50, wherein said genotype comprises a nucleotide sequence having atleast 95% sequence identity to SEQ ID NO: 27 and SEQ ID NO:
 30. 52. Thecorn plant of claim 50, wherein said genotype comprises a nucleotidesequence having at least 95% sequence identity to SEQ ID NO: 28 and SEQID NO:
 31. 53. The corn plant of claim 50, wherein the genotypecomprises a nucleotide sequence having 1, 2, 3, 4, or 5 nucleotidechanges in one of SEQ ID NO: 26 or SEQ ID NO:
 27. 54. A corn plantexpressing an insecticidal protein from a fern and exhibiting a meannode-injury root rating of at least about 0.15 against corn root worm incombination with a bacterial protein exhibiting insecticidal activityagainst corn root worm and optionally expressing a dsRNA that targets anendogenous gene of corn rootworm, wherein the insecticidal protein, thebacterial insecticidal protein and the dsRNA display different mode ofactions.
 55. A corn plant expressing three distinct modes of actionsagainst western corn rootworm, wherein at least two modes are due toinsecticidal proteins not derived from a bacteria.